Feruloyl esterases synthesize butyl hydroxycinnamates, molecules possessing interesting biological properties, nonetheless, they exhibit a low stability under synthesis conditions in organic solvents, restricting its use. To enhance its operational stability in synthesis, we immobilized type A feruloyl esterase from Aspergillus niger (AnFAEA) using several carrier-bound and carrier-free strategies. The most active biocatalysts were: 1) AnFAEA immobilized on epoxy-activated carriers (protein load of 0.6 mgenzyme x mg-1carrier) that recovered 91 % of the initial hydrolytic activity, and 2) AnFAEA aggregated and cross-linked in the presence of 5 mg of BSA and 15 mM of glutaraldehyde (AnFAEA-amino-CLEAs), which exhibited 385 % of its initial hydrolytic activity; both using 4-nitrophenyl butyrate as substrate. The AnFAEA-amino-CLEAs were 12.7 times more thermostable at 60 °C than the AnFAEA immobilized on epoxy-activated carrier, thus AnFAEA-amino-CLEAs were selected for further characterization. Interestingly, during methyl sinapate hydrolysis (pH 7.2 and 30 °C), AnFAEA-amino-CLEAs KM was 15 % higher, while during butyl sinapate synthesis the KM was reduced in 63 %, both compared with the soluble enzyme. The direct esterification of butyl sinapate at solvent free conditions using sinapic acid 50 mM, reached 95 % conversion after 24 h employing AnFAEA-amino-CLEAs, which could be used for 10 cycles without significant activity losses, demonstrating their outstanding operational stability.

中文翻译：

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黑曲霉A型阿魏酸酯酶的无载体结合和无载体固定化：寻找可操作稳定的异质生物催化剂，用于合成羟基肉桂酸丁酯。

阿魏酸酯酶合成羟基肉桂酸丁酯，该分子具有令人感兴趣的生物学特性，尽管如此，它们在有机溶剂中的合成条件下仍显示出较低的稳定性，从而限制了其用途。为了增强其在合成中的操作稳定性，我们使用几种与载体结合和无载体的策略固定了来自黑曲霉（AnFAEA）的A型阿魏酸酯酶。活性最高的生物催化剂是：1）AnFAEA固定在环氧活化的载体上（蛋白质负载为0.6 mgenzyme x mg-1载体），可恢复初始水解活性的91％； 2）AnFAEA在5存在下聚集并交联毫克的牛血清白蛋白和15毫米的戊二醛（AnFAEA-氨基-CLEAs），表现出其初始水解活性的385％；两者均使用4-硝基苯基丁酸酯作为底物。AnFAEA-氨基-CLEA为12。在60°C时的热稳定性是固定在环氧活化载体上的AnFAEA的7倍，因此选择了AnFAEA-氨基-CLEA用于进一步表征。有趣的是，在芥子酸甲酯水解过程中（pH 7.2和30°C），AnFAEA-氨基-CLEAs KM比可溶酶高15％，而在芥子酸丁酯合成过程中，KM降低63％。在无溶剂条件下，使用芥子酸50 mM在无溶剂条件下直接将正庚酸丁酯直接酯化，使用AnFAEA-氨基-CLEAs在24 h后可达到95％的转化率，可用于10个循环而没有明显的活性损失，证明了其出色的操作稳定性。与可溶性酶相比，在芥子酸甲酯水解（pH 7.2和30°C）期间，AnFAEA-氨基-CLEAs KM升高15％，而在芥子酸丁酯合成期间，KM降低63％。在无溶剂条件下，使用芥子酸50 mM在无溶剂条件下直接将正庚酸丁酯直接酯化，使用AnFAEA-氨基-CLEAs在24 h后可达到95％的转化率，可用于10个循环而没有明显的活性损失，证明了其出色的操作稳定性。与可溶性酶相比，在芥子酸甲酯水解（pH 7.2和30°C）期间，AnFAEA-氨基-CLEAs KM升高15％，而在芥子酸丁酯合成期间，KM降低63％。在无溶剂条件下，使用芥子酸50 mM在无溶剂条件下直接将正庚酸丁酯直接酯化，使用AnFAEA-氨基-CLEAs在24 h后可达到95％的转化率，可用于10个循环而没有明显的活性损失，证明了其出色的操作稳定性。